This invention is in the field of assays, including immunoassays, for the detection of selected analytes in a fluid sample.
As used herein, the term xe2x80x9cligand-receptor assayxe2x80x9d refers to an assay for an analyte which may be detected by the formation of a complex between a ligand and a ligand receptor which is capable of specific interaction with that ligand. The ligand may be the analyte itself or a substance which, if detected, can be used to infer the presence of the analyte in a sample. In the context of the present invention, the term xe2x80x9cligandxe2x80x9d, includes haptens, hormones, antigens, antibodies, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), metabolites of the aforementioned materials and other substances of either natural or synthetic origin which may be of diagnostic interest and have a specific binding partner therefor, i.e., the ligand receptor of the ligand-receptor assay. In the context of the present invention the term xe2x80x9cligand receptorxe2x80x9d includes materials for which there is a specific binding partner, i.e., the ligand of the ligand-receptor assay. Those skilled in the art will appreciate that the analyte of interest is a member of a specific binding pair and may be either a ligand receptor or a ligand depending upon assay design.
Ligand-receptor assays are generally useful for the in vitro determination of the presence and concentration of ligands in body fluids, food products, animal fluids, and environmental samples. For example, the determination of specific hormones, proteins, therapeutic drugs, and toxic drugs in human blood or urine has significantly improved the medical diagnosis of the human condition. There is a continuing need for simple, rapid, non-instrumental assays for the qualitative, semi-quantitative, and quantitative determination of such ligands in a sample. In many situations, such assays need to be simple enough to be performed and interpreted by non-technical users. In addition, there has existed an unmet need to determine the presence and concentration of multiple ligands in a single assay. For example, the need exists for a rapid analytical tool to determine, in the emergency rooms of hospitals, the presence of the multiple drugs of abuse.
Ligand-receptor assays rely on the binding of ligands by ligand receptors to determine the concentration of ligands in a sample. Ligand-receptor assays can be described as either competitive or non-competitive. Non-competitive assays generally utilize ligand receptors in substantial excess over the concentration of ligand to be determined in the assay. Sandwich assays, in which the ligand is detected by binding to two ligand receptors, one ligand receptor labeled to permit detection and the second ligand receptor frequently bound to a solid phase to facilitate separation from unbound reagents, such as unbound labeled first ligand receptor, are examples of non-competitive assays. Competitive assays generally involve ligand from the sample, a ligand analogue labeled to permit detection, and the competition of these species for a limited number of binding sites provided by the ligand receptor. Those skilled in the art will appreciate that many variations of this basic competitive situation have been previously described and will not be discussed in detail herein except where pertinent to the general objectives of this invention. Examples of ligands which are commonly measured by competitive ligand-receptor assays include haptens, hormones and proteins. Antibodies that can bind these classes of ligands are frequently used in these assays as ligand receptors.
Competitive ligand-receptor assays can be further described as being either homogeneous or heterogeneous. In homogeneous assays all of the reactants participating in the competition are mixed together and the quantity of ligand is determined by its effect on the extent of binding between ligand receptor and labeled ligand analogue. The signal observed is modulated by the extent of this binding and can be related to the amount of ligand in the sample. U.S. Pat. No. 3,817,837 describes such a homogeneous, competitive immunoassay in which the labeled ligand analogue is a ligand-enzyme conjugate and the ligand receptor is an antibody capable of binding to either the ligand or the ligand analogue. The binding of the antibody to the ligand-enzyme conjugate decreases the activity of the enzyme relative to the activity observed when the enzyme is in the unbound state. Due to competition between unbound ligand and ligand-enzyme conjugate for antibody binding sites, as the ligand concentration increases the amount of unbound ligand-enzyme conjugate increases and thereby increases the observed signal. The product of the enzyme reaction may then be measured kinetically using a spectrophotometer.
In general, homogeneous assay systems require both an instrument to read the result and calibration of the observed signal by separate tests with samples containing known concentrations of ligand. The development of homogeneous assays has dominated competitive assay research and has resulted in several commercially available systems. Such systems are not, however, capable of providing results for the determination of multiple ligands in a sample in a single-test format not requiring complex instrumentation.
Heterogeneous, competitive ligand-receptor assays require a separation of bound labeled ligand or receptor from the free labeled ligand or receptor and a measurement of either the bound or the free fraction. Methods for performing such assays are described in U.S. Pat. Nos. 3,654,090, 4,298,685, and 4,506,009. Such methods, however, are not capable of providing semi-quantitative or quantitative results for the determination of ligands in a sample without using additional tests to calibrate the assay response.
The need for ligand-receptor assays that can be performed without the use of complex instrumentation has led to the development of immunoassays that are simple to perform and result in a response that can be visually interpreted. U.S. Pat. Nos. 4,125,372, 4,200,690, 4,246,339, 4,366,241, 4,446,232, 4,477,576, 4,496,654, 4,632,901, 4,727,019, and 4,740,468 describe devices and methods for ligand-receptor assays that develop colored responses for visual interpretation of the results. While such devices provide simple formats for the visual interpretation of assay results, only the presence or absence of ligand can be determined; semi-quantitative or quantitative determinations using these methods require that separate tests utilizing standards of known concentration be performed to establish the relationship between the observed response and the concentration of ligand.
Employing the techniques described for competitive ligand-receptor assays, the intensity of the resulting color is inversely related to the concentration of ligand in the sample such that assay results that are more intense in color than the reference are interpreted to mean that the sample contained ligand at a lower concentration than that represented by the concentration by the reference. A serious drawback, however, to the widespread utilization of such visually interpreted competitive ligand-receptor assays has been this inverse relationship between intensity of the developed signal and sample ligand concentration. This relationship provides that a sample with a low concentration of ligand will produce a large signal in the assay; and conversely a sample with a high concentration of ligand will produce a small signal in the assay. A further disadvantage of such assays is that if the requirement is for a single test to simultaneously determine multiple ligands each of which must be assigned a semi-quantitative value and each of which has specific individual concentration targets, then individual specific reference zones would have to be provided for each ligand to be determined. Under such circumstances, a test for multiple ligands becomes difficult to produce and complex to interpret.
Another prior art approach, a non-competitive immunochromatographic assay, is described in U.S. Pat. Nos. 4,168,146 and 4,435,504. This assay provides a method for quantitatively determining the presence of a single analyte in a sample in a visually interpreted immunoassay but does not permit the assay of multiple analytes without employing multiple devices. Furthermore, in practice this method is restricted to ligands whose sample concentrations are high relative to ligands that are commonly determined by competitive assay technology. Accordingly, this type of approach is of limited utility. Clearly, there is an unmet need for a ligand-receptor assay capable of determining the presence of a multiplicity of ligands in a sample and concurrently providing individualized semiquantitative results for each ligand. Furthermore, such an assay should produce such results in a format that is simple enough for an non-technical user to correctly perform and interpret. In addition there is a need for broadly applicable quantitative assay methods that are easily performed and interpreted. The inventive methods of this invention and those described in U.S. Pat. Nos. 5,028,535 and 5,089,391 meet these requirements.
Methods to prepare monoclonal antibodies to ligands which, by themselves, do not generate an immunological response are well known to those skilled in the art. The ligand, or an analogue thereof, is generally coupled, chemically, to a carrier molecule, e.g., a protein, peptide, or other polymer, to form an immunogen (one example of a ligand analogue conjugate as defined herein) which elicits an immunological response. Antibodies are thus raised to the surface of the carrier molecule onto which is coupled the ligand. The selection or screening of antibodies is then performed to choose the antibody which best fulfills the intended use of the antibody. The screening of antibodies is well known to those skilled in the art and is generally performed by binding a ligand-carrier conjugate to a solid phase, allowing the raised antibody to bind to the ligand-carrier conjugate and detecting the presence of the bound antibody with a labelled anti-antibody conjugate. An inherent problem with the generation and screening of antibodies is the difficulty in determining the location of binding of the antibody to the ligand, i.e., the binding site; that is, it is not clear which portion of the ligand analogue is bound by the antibody. This can result in the selection of antibodies which possess a very small but definite affinity to the carrier molecule, or to the chemical structure (herein called the xe2x80x9clinkage sitexe2x80x9d) which attaches the ligand analogue to carrier molecule. Such an antibody, will thus bind (an occurrence known as crosstalk) to other, or uncomplementary, carrier molecule-ligand complexes having such a linkage, and produce false positive results when such other complexes are present in a test.
The crosstalk problem has previously been dealt with by using different linkage chemistries for attaching the ligand to the carrier molecule, and for screening the ligand-carrier conjugate; for example, see Van Weemen and Schuurs, The influence of heterologous combinations of antiserum and enzyme-labeled estrogen on the characteristics of estrogen enzyme-immunoassays, Immunochemistry, 12, 667 (1975). In developing ligand receptor assays for a multitude of ligands, however, creating such different chemical structures for multiple ligands can be very time consuming and expensive. In addition, this approach to developing ligand receptors is not guaranteed to be successful because the similarities of linkage chemistries, in general, make all linkage chemistries similar to a certain extent. Also, if one is attempting to prepare ligand receptors with a high degree of specificity for two molecules with very similar structures, one may be limited to the numbers of ligand analogues which can be synthesized.
The development of a ligand receptor assay capable of determining the presence of a multiplicity of ligands requires that the ligand receptors not have a substantial affinity for uncomplementary ligand analogue conjugates. A substantial affinity of a ligand receptor for an uncomplementary ligand analogue conjugate in a multi-analyte assay results in false positive results and renders the assay useless.
The present invention is related to reagents, and method of their use, which reduce or prevent the crosstalk or the undesirable interactions between ligand receptors and uncomplementary ligand analogue conjugates. The reagents, or crosstalk inhibitors, described herein mimic the chemical structure which links the ligand or ligand analogue to the carrier molecule. The extent to which the crosstalk inhibitor must or must not resemble the chemical structure of the ligand analogue linkage chemistry depends on the affinity that the ligand receptor possesses for the linkage chemistry. Thus, the reagents described herein allow the simultaneous determination of the presence of a multiplicity of ligands in a sample by inhibiting or reducing the low affinity interactions of the ligand receptors for the uncomplementary ligand-carrier molecule conjugate or other uncomplementary ligand analogue conjugates.
The present invention is directed to ligand receptor assays in which the presence of a multiplicity of ligands are measured in a single determination. In particular, the present invention relates to the preparation and use of reagents as crosstalk inhibitors in ligand receptor assays. The crosstalk inhibitors resemble the chemical structure (or linkage site) which links the ligand analogue to the carrier molecule of a ligand analogue conjugate. Thus, the crosstalk inhibitors reduce or prevent the crosstalk, i.e., the undesirable interactions between ligand receptors and uncomplementary ligand analogue conjugates.
More particularly, this invention relates to methods and reagents for the simultaneous determination of more than one target ligand in a single test format. The inventive assays described herein involve the use of crosstalk inhibitors in simultaneous multi-ligand assays. The crosstalk inhibitors resemble the chemical structure which links a ligand, or an analogue thereof, to, e.g., a signal development element of a ligand analogue conjugate, and thereby reduce or prevent undesirable interactions between ligand receptors and uncomplementary ligand analogue conjugates.
The crosstalk inhibitor is normally contained in the reaction mixture. It does not compete significantly with the complementary ligand analogue conjugate for ligand receptor binding sites in the reaction mixture. Once the binding events in the reaction mixture reflect the amounts of target ligands in the sample (also expressed as the reaction mixture reaching substantial equilibrium binding), the reaction mixture may be contacted with a terminal solid phase onto which is immobilized ligand receptor. In the absence of crosstalk inhibitor, the concentration of ligand receptor on the solid phase is such that the unbound ligand analogue conjugate can bind monovalently or multivalently to the solid phase ligand receptor or receptors. This ability of the ligand analogue conjugate to bind multivalently to the solid phase ligand receptor amplifies low affinity interactions because the effective affinity is the product of the individual affinities of the monovalent interactions. The result is that ligand receptors on the solid phase, which have low affinities for the linkage chemistries of the ligand analogue conjugates, can bind multivalently to uncomplementary ligand analogue conjugates. The product of such multivalent binding is large enough to detect a binding of uncomplementary ligand analogue conjugate to the solid phase ligand receptor as a result of the assay. Such detection provides a false positive result.
The crosstalk inhibitor, which resembles the linkage chemistry of the ligand analogue conjugates, competes with the linkage chemistry of the ligand analogue conjugates for binding to the terminal solid phase ligand receptor. With the proper crosstalk inhibitor and crosstalk inhibitor concentration, the competition is shifted toward binding of the crosstalk inhibitor and not of the uncomplementary ligand analogue conjugates. The crosstalk inhibitor should compete very poorly with the complementary ligand analogue conjugate for the solid phase ligand receptor because the affinity of the ligand receptor for the complementary ligand analogue conjugate is much higher.
How closely the chemical structure of the crosstalk inhibitor must resemble the linkage chemistry of the ligand analogue depends on the affinity of the ligand receptor for the uncomplementary ligand analogue conjugate. The crosstalk inhibitor may be free in solution or bound to a protein or polymer. When the crosstalk inhibitor is attached to a protein or polymer, it can bind multivalently to the solid phase ligand receptor as can the ligand analogue conjugate. Thus, the multivalent crosstalk inhibitor can better compete with the uncomplementary ligand analogue conjugate than the monovalent crosstalk inhibitor.
Definitions
In interpreting the claims and specification, the following terms shall have the meanings set forth below.
Complementary Ligand Analogue Conjugatexe2x80x94This ligand analogue conjugate binds to its intended ligand receptor at the binding site. By intended ligand receptor is meant, e.g., a ligand receptor produced by standard immunological techniques to the conjugate itself, or to its equivalent analogue. Those of ordinary skill in the art will recognize the scope of this term when used to describe non-antibody ligands, and their conjugates. As an example, in the antibody-type ligand area, a complementary ligand analogue conjugate will have a higher affinity for its intended ligand receptor of at least 105 Mxe2x88x921 (for monovalent binding), and preferably higher, and an uncomplementary ligand analogue conjugate (see below) will have a lower affinity (for monovalent binding) for this ligand receptor than the complementary ligand analogue conjugate.
Complementary Ligand Receptorxe2x80x94This ligand receptor binds its intended ligand analogue conjugate at the binding site.
Crosstalkxe2x80x94The binding of the uncomplementary ligand conjugate to the ligand receptor, e.g., at the linkage site of the conjugate. Also, the binding of an uncomplementary ligand receptor conjugate to a ligand analogue.
Crosstalk inhibitorxe2x80x94The crosstalk inhibitor is an analogue or analogues of the linkage chemistry, at a linkage site, which is used to attach ligand analogue to a protein, polypeptide, polymer or molecular complex, and reduces or prevents the binding of an uncomplementary ligand analogue conjugate to ligand receptor or the uncomplementary ligand receptor conjugate to ligand analogue. It does not interfere significantly with binding of a complementary ligand analogue conjugate at its binding site to a ligand receptor. The crosstalk inhibitor can be free in solution, alone or bound to itself as a dimer or multimer, or it can be attached to proteins, polypeptides, polymers or molecular complexes.
Ligandxe2x80x94Binding partner to ligand receptor.
Ligand Analoguexe2x80x94A chemical derivative of the ligand which may be attached either covalently or noncovalently to other species, for example, to the signal development element. Ligand analogue and ligand may be the same, and both are capable of binding to ligand receptor.
Ligand Analogue Conjugatexe2x80x94A conjugate of a ligand analogue and a signal development element, a protein, polypeptide, or polymer. When a non-signal development element (the ligand analogue conjugate of which is herein termed a ligand analogue construct) is used, its presence in an assay as a binding partner may be detected by standard procedure, e.g., using methodology commonly used in sandwich assays, such as a labelled antibody to the ligand analogue construct.
Ligand Receptorxe2x80x94Receptor capable of binding ligand, typically an antibody, but which may be another ligand, depending on assay design.
Binding domainxe2x80x94shall refer to the molecular structure associated with that portion of a receptor that binds ligand. More particularly, the binding domain may refer to a polypeptide, natural or synthetic, or nucleic acid encoding such a polypeptide, whose amino acid sequence represents a specific region of a protein, said domain, either alone or in combination with other domains, exhibiting binding characteristics which are the same or similar to those of a desired ligand/receptor binding pair. Neither the specific sequences nor the specific boundries of such domains are critical, so long as binding activity is exhibited. Likewise, used in this context, binding characteristics necessasrily includes a range of affinities, avidities and specificities, and combinations thereof, so long as binding activity is exhibited.
Linking groupxe2x80x94shall mean the xe2x80x9cchemical armxe2x80x9d between the protein, polypeptide or label and a drug or drug derivative. As one skilled in the art will recognize, to accomplish the requisite chemical structure, each of the reactants must contain the necessary reactive groups. Representative combinations of such groups are amino with carboxyl to form amide linkages, or carboxy with hydroxy to form ester linkages or amino with alkyl halides to form alkylamino linkages, or thiols with thiols to form disulfides, or thiols with maleimides or alkkylhalides to form thioethers. Obviously, hydroxyl, carboxyl, amino and other functionalities, where not present may be introduced by known methods. Likewise, as those skilled in the art will recognize, a wide variety of linking groups may be employed. The structure of the linkage should be a stable covalent linkage formed to bind the drug or drug derivative to the protein, polypeptide or label. In some cases the linking group may be designed to be either hydrophilic or hydrophobic in order to enhance the desired binding characteristics of the ligand and the receptor. The covalent linkages should be stable relative to the solution conditions under which the ligand and linking group are subjected. Generally preferred linking groups will be from 1-20 carbons and 0-10 heterocarbons (NH, O, S) and may be branched or straight chain. Without limiting the foregoing, it should be obvious to one skilled in the art that only combinations of atoms which are chemically compatible comprise the linking group. For example, amide, ester, thioether, thioester, keto, hydroxyl, carboxyl, ether groups in combinations with carbon-carbon bonds are acceptable examples of chemically compatible linking groups. Other chemically compatible compounds which may comprise the linking group are set forth in this Definition section and hereby are incorporated by reference.
Hydrocarbylxe2x80x94shall refer to an organic radical comprised of carbon chains to which hydrogen and other elements are attached. The term includes alkyl, alkenyl, alkynyl and aryl groups, groups which have a mixture of saturated and unsaturated bonds, carbocyclic rings and includes combinations of such groups. It may refer to straight-chain, branched-chain cyclic structures or combinations thereof.
arylxe2x80x94shall refer to aromatic groups which have at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted.
Carbocyclic aryl groupsxe2x80x94shall refer to groups wherein the ring atoms on the aromatic ring are carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups and optionally substituted naphthyl groups.
Monocyclic carbocyclic arylxe2x80x94shall refer to optionally substituted phenyl, being preferably phenyl or phenyl substituted by one to three substituents, such being advantageously lower alkyl, hydroxy, lower alkoxy, lower alkanoyloxy, halogen, cyano, trihalomethyl, lower acylamino, lower amino or lower alkoxycarbonyl.
Optionally substituted naphthylxe2x80x94shall refer to 1- or 2-naphthyl or 1- or 2-naphthyl preferably substituted by lower alkyl, lower alkoxy or halogen.
Heterocyclic aryl groupsxe2x80x94shall refer to groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl, and the like, all optionally substituted.
Optionally substituted furanylxe2x80x94shall refer to 2- or 3-furanyl or 2- or 3-furanyl preferably substituted by lower alkyl or halogen.
Optionally substituted pyridylxe2x80x94shall refer to 2-, 3- or 4-pyridyl or 2-, 3- or 4-pyridyl preferably substituted by lower alkyl or halogen.
Optionally substituted thienylxe2x80x94shall refer to 2- or 3-thienyl, or 2- or 3-thienyl preferably substituted by lower alkyl or halogen.
biarylxe2x80x94shall refer to phenyl substituted by carbocyclic aryl or heterocyclic aryl as defined herein, ortho, meta or para to the point of attachment of the phenyl ring, advantageously para; biaryl is also represented as the xe2x80x94C6H4xe2x80x94Ar substituent where Ar is aryl.
aralkylxe2x80x94shall refer to an alkyl group substituted with an aryl group. Suitable aralkyl groups include benzyl, picolyl, and the like, and may be optionally substituted.
lowerxe2x80x94referred to herein in connection with organic radicals or compounds respectively defines such with up to and including 7, preferably up to and including 4 and advantageously one or two carbon atoms. Such groups may be straight chain or branched.
The terms (a) xe2x80x9calkyl aminoxe2x80x9d, (b) xe2x80x9carylaminoxe2x80x9d, and (c) xe2x80x9caralkylaminoxe2x80x9d, respectively, shall refer to the groups xe2x80x94NRRxe2x80x2 wherein respectively, (a) R is alkyl and Rxe2x80x2 is hydrogen or alkyl; (b) R is aryl and Rxe2x80x2 is hydrogen or aryl, and (c) R is aralkyl and Rxe2x80x2 is hydrogen or aralkyl.
The term xe2x80x9cacylxe2x80x9d shall refer to hydrocarbyl-COxe2x80x94 or HCOxe2x80x94.
The terms xe2x80x9cacylaminoxe2x80x9d refers to RCONCR)xe2x80x94 and (RCO2Nxe2x80x94 respectively, wherein each R is independently hydrogen or hydrocarbyl.
The term xe2x80x9chydrocarbyloxycarbonyloxyxe2x80x9d shall refer to the group ROC(O)Oxe2x80x94 wherein R is hydrocarbyl.
The term xe2x80x9clower carboalkoxymethylxe2x80x9d or xe2x80x9clower hydrocarbyloxycarbonymethylxe2x80x9d refers to hydrocarbyl-OC(O)CH2xe2x80x94 with the hydrocarbyl group containing ten or less carbon atoms.
The term xe2x80x9ccarbonylxe2x80x9d refers to xe2x80x94C(O)xe2x80x94. The term xe2x80x9ccarboxamidexe2x80x9d or xe2x80x9ccarboxamidoxe2x80x9d refers to xe2x80x94CONR2 wherein each R is independently hydrogen or hydrocarbyl.
The term xe2x80x9clower hydrocarbylxe2x80x9d refers to any hydrocarbyl group of ten or less carbon atoms.
The term xe2x80x9calkylxe2x80x9d refers to saturated aliphatic groups including straight-chain, branched chain and cyclic groups.
The term xe2x80x9calkenylxe2x80x9d refers to unsaturated hydrocarbyl groups which contain at least one carbon-carbon double bond and includes straight-chain, branched-chain and cyclic groups.
The term xe2x80x9calkynylxe2x80x9d refers to unsaturated hydrocarbyl groups which contain at least one carbon-carbon triple bond and includes straight-chain, branched-chain and cyclic groups.
The term xe2x80x9chydrocarbyloxycarbonylaminoxe2x80x9d refers to a urethane, hydrocarbyl-Oxe2x80x94CONRxe2x80x94 wherein R is H or hydrocarbyl and wherein each hydrocarbyl is independently selected. The term xe2x80x9cdi(hydrocarbyloxycarbonyl)aminoxe2x80x9d refers to (hydrocarbyl-Oxe2x80x94CO)2Nxe2x80x94 wherein each hydrocarbyl is independently selected.
The term xe2x80x9chydrocarbylaminoxe2x80x9d refers to xe2x80x94NRRxe2x80x2 wherein R is hydrocarbyl and Rxe2x80x2 is independently selected hydrocarbyl or hydrogen.
The term xe2x80x9cmercaptoxe2x80x9d refers to SH or a tautomeric form.
The term xe2x80x9cmethinexe2x80x9d refers to 
The term xe2x80x9cmethylenexe2x80x9d refers to xe2x80x94CH2xe2x80x94,
The term xe2x80x9calkylenexe2x80x9d refers to a divalent straight chain or branched chain saturated aliphatic radical.
The term xe2x80x9coxyxe2x80x9d refers to xe2x80x94Oxe2x80x94 (oxygen).
The term xe2x80x9cthioxe2x80x9d refers to xe2x80x94Sxe2x80x94 (sulfur).
Disulfidexe2x80x94refers to xe2x80x94Sxe2x80x94Sxe2x80x94.
Thioetherxe2x80x94refers to Cxe2x80x94Sxe2x80x94C.
Esterxe2x80x94refers to 
Reaction Mixturexe2x80x94In a competitive immunoassay, the mixture of sample suspected of containing the target ligand and the assay reagents that participate in the competitive binding reactions.
Signal Development Elementxe2x80x94The element of the ligand analogue conjugate, e.g., an enzyme, which, in conjunction with the signal development phase, develops a detectable signal. Signal Development Phasexe2x80x94The phase containing the materials enabling the signal development element to develop signal, e.g., an enzyme substrate solution.
Signal Development Systemxe2x80x94A system which includes those reagents necessary to detect binding of a binding pair.
Terminal Solid Phasexe2x80x94The solid phase upon which the signal is finally developed during signal development.
Threshold Concentrationxe2x80x94The concentration of ligand in a sample which results in the first detectable signal development. A threshold concentration is a concentration reference point.
Uncomplementary Ligand Analogue Conjugatexe2x80x94This ligand analogue conjugate binds to ligand receptors other than its intended ligand receptor, at its linkage site or another site, but not at its binding site.
Uncomplementary Ligand Receptorxe2x80x94This ligand receptor binds an unintended ligand analogue conjugate at the linkage site. (A particular ligand receptor may be both a complementary and an uncomplementary ligand receptor dependent upon the ligand analogue conjugate in issue.)
Thus, in a first aspect, the invention features an assay for detecting the amount or presence of target ligand in a sample. The assay includes a ligand analogue conjugate having a linkage site and a binding site, a ligand receptor, and a sample. The assay includes the steps of providing at least one crosstalk inhibitor. This inhibitor, under assay conditions, competes with the linkage site of the ligand analogue conjugate for binding to the ligand receptor, and does not compete with the binding site of the ligand analogue conjugate for binding to the ligand receptor. In the invention, the assay is performed for the target ligand in the presence of a sufficient amount of the crosstalk inhibitor to reduce the amount of binding of the linkage site of the uncomplementary ligand analogue conjugate to the ligand receptor.
By xe2x80x9cnot competexe2x80x9d is meant that while the crosstalk inhibitor may compete to some extent with binding of the binding site of the ligand analogue conjugate and the ligand receptor, that competition does not prevent detection of a positive interaction between the ligand analogue conjugate and ligand receptor. That is, the amount of competition is not significant in the assay, since the assay still provides a detectable true positive result, with fewer false positive results. It is important in this invention that such a true positive interaction is still observable, and that the number or amount of false positive results is reduced. Examples of such non-competition are provided below in the examples. Those of ordinary skill in this art will recognize that these examples show and elaborate on the meaning of the term xe2x80x9cnot competexe2x80x9d. Thus, while a positive response may be reduced by 50% or more, it is important only that this result is detectable, and that the false positive response is reduced to a low level, preferably to an undetectable level. A crosstalk inhibitor which produces such a result is useful in this invention and is said to xe2x80x9cnot competexe2x80x9d with the binding site of the ligand analogue conjugate for the ligand receptor, but to xe2x80x9ccompetexe2x80x9d with the linkage site of the ligand analogue conjugate for the ligand receptor.
In a related aspect, the invention features an assay for detecting the amount or presence of a plurality of target ligands in a sample. This assay includes a plurality of ligand analogue conjugates corresponding to the plurality of target ligands. Each ligand analogue conjugate has a linkage site and a binding site; the binding site of each ligand analogue conjugate is generally different from the binding site of other ligand analogues conjugates. The linkage site of each ligand analogue conjugate may be the same or different. The assay also includes a plurality of ligand receptors. Each ligand receptor binds with only one target ligand of the plurality of target ligands, and not with the other target ligand, with each ligand receptor binding with a different target ligand. The assay also includes a sample. In the method, at least one crosstalk inhibitor is provided. This crosstalk inhibitor, under assay conditions, competes with the linkage site of at least one ligand analogue conjugate for binding to at least one ligand receptor, and does not compete with any of the binding sites of the plurality of ligand analogue conjugates for binding to the plurality of ligand receptors. Finally, in the method, an assay is performed for the target ligands in the presence of a sufficient amount of the crosstalk inhibitor to reduce the amount of binding to the linkage site of at least one ligand analogue conjugate to at least one uncomplementary ligand receptor.
In another aspect, the invention features a method for identifying a crosstalk inhibitor useful in an assay which detects the presence or amount of target ligand in a sample. The assay includes a complementary ligand analogue conjugate having a linkage site and a binding site, a ligand receptor and a sample. In the method, a potential crosstalk inhibitor is provided and a test performed which includes essentially the following components: the ligand analogue conjugate, an uncomplementary ligand receptor, and a signal development system. In this test, a positive result is attained in the absence of a crosstalk inhibitor (i.e., a false positive result is attained). This test is also performed in the presence of the potential crosstalk inhibitor. A reduced false positive result in the presence of crosstalk inhibitor as compared to the false positive result obtained in the absence of the crosstalk inhibitor indicates that the potential crosstalk inhibitor is a useful crosstalk inhibitor. Such a reduced false positive result is generally a result of competition between the crosstalk inhibitor and the uncomplementary ligand analogue conjugate for the ligand receptor. (The term xe2x80x9ccompetitionxe2x80x9d is herein defined as the phenomenon observed to occur between crosstalk inhibitors, in the examples provided below, and the ligand analogue conjugates for the ligand receptor.)
In a preferred embodiment, the potential crosstalk inhibitor is also tested in an assay which provides a true positive result to ensure that such a true positive result is still attained in the presence of the inhibitor. The inhibitor is useful when it allows detection of such a positive result and reduces the level of false positive results. By varying the concentration of the inhibitor, or by varying its valency (e.g., by providing inhibitor bound multivalently to BSA) the use of such an inhibitor can be optimized. Examples of such variations are provided below.
In yet another aspect, the invention features a crosstalk inhibitor useful in an assay for detecting the amount or presence of an analyte in a sample. The assay includes at least one ligand analogue conjugate having a linkage site and a binding site, at least one ligand receptor and a sample. The crosstalk inhibitor competes with the linkage site of the ligand analogue conjugate for binding to the ligand receptor and does not compete with the binding site of the ligand analogue conjugate for binding to the ligand receptor.
The drawings will first briefly be described.